The production of proteins in genetically engineered host cells is practiced since many years. Prokaryotic (bacterial) cells and eukaryotic cells such as yeast cells and mammalian cells are used for the production of therapeutic proteins or industrially useful proteins such as enzymes for the production of food, detergents and the like. In particular, protein production in eukaryotic, in particular mammalian cells is an important tool in numerous scientific and commercial areas. For example, the proteins expressed in and purified from mammalian cell systems are routinely needed for life science research and development. In the field of biomedicine, proteins for human therapy, vaccination or diagnostic applications are typically produced in mammalian cells. Gene cloning, protein engineering, biochemical and biophysical characterization of proteins also require the use of gene expression in mammalian cells. Other applications in widespread use involve screening of libraries of chemical compounds in drug discovery, and the development of cell-based biosensors.
A vast number of expression systems are used to produce recombinant proteins, ranging from cell free systems to cell based systems. Presently, due to technical limitations associated with cell free expression systems, cell based systems are more commonly used for recombinant protein expression. Cell based expression systems include those utilizing bacteria, yeast, insect cells or mammalian cells as hosts. The majority of these systems utilize inducible expression. For example, in a recent international collaboration to produce and purify over 10,000 recombinant proteins for use in structural biology, well over 90% of these proteins were produced using some form of inducible expression system (Nature Methods 5, 135-146).
Typical examples of expression systems and purification processes are described in WO 95/02049. Host cells are lysed in suspension culture, the lysate is applied to a filter such that the compound of interest, a nucleic acid, is bound by the filter while the remaining lysate is separated from the compound of interest. A similar disclosure is presented in WO 2008/066858, with the exception that host cells containing the compound of interest, a nucleic acid, are first lysed in suspension such that their outer membrane is destroyed and then lysed to destroy the nuclear membrane so as to release the nucleic acid from the nucleus. Thus, this PCT-application provides, so to say, a step-wise lysis of host cells in suspension to finally release the compound of interest. Another quite similar approach is disclosed in WO 03/070898. Specifically, a heterologous group of cells are lysed to release DNA from a first cell type so as to then collect unlysed cells which are subsequently subjected to lysis. This step-wise approach allows the isolation of DNA from different types of cells. WO 2009/157680 is a typical example for the isolation of DNA from bacterial host cells. These host cells are lysed to release their DNA on a hydrogel column in order to have it available for further steps such as genetic engineering. Prior to applying the host cells to the hydrogel column, these host cells are lysed in suspension culture. An almost identical approach is presented in U.S. 2009/0325269, with the exception that the host cells are repeatedly lysed in an apparatus so as to quantitatively release their DNA which is the desired target material. A further example for the isolation of DNA from host cells, in particular bacterial host cells, is described in U.S. Pat. No. 5,834,303. Bacterial host cells are lysed to release their DNA which is then subject to column chromatography in order to purify the DNA. All these documents have in common that they provide processes for recovering DNA, preferably from bacterial host cells or mammalian cells. However, none of these documents aims at the recovery of proteins, let alone viruses. This is so because for proteins and in particular for viruses other well-established processes are commonly applied such as homogenization, freeze-thawing or lysis in suspension followed by various filtration steps.
Eukaryotic cells such as mammalian cells and avian cells are frequently used for the production of viruses. The arising of new threats (avian flu, west nile virus, anthrax, pox disease, etc.) as well as the development of gene therapy has increased the need for producing and purifying poxviruses for prophylactic or therapeutic purposes, especially for viruses as vaccines. This is notably the case for the Modified Vaccinia Virus Ankara (MVA). This poxvirus which was initially used for vaccinating immunodeficient patients against smallpox is now also used as a vector for gene therapy purposes. MVA carrying the gene coding for Human Papilloma Virus (HPV) or Human Immunodeficiency Virus (HIV) antigens is also used as a vector for the therapeutic treatment of ovarian carcinoma and HIV, respectively.
Poxviruses are a group of complex enveloped viruses that distinguish them principally by their unusual morphology, their large DNA genome and their cytoplasmic site of replication. The genome of several members of poxviridae, including the Copenhagen vaccinia virus (W) strain (Goebel et al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401) and the modified vaccinia virus Ankara (MVA) strain (Antoine et al., 1998, Virol. 244, 365-396), have been mapped and sequenced. W has a double-stranded DNA genome of about 192 kb coding for about 200 proteins of which approximately 100 are involved in virus assembly. MVA is a highly attenuated vaccinia virus strain generated by more than 500 serial passages of the Ankara strain of vaccinia virus on chicken embryo fibroblasts (Mayr et al., 1975, Infection 3, 6-14 Swiss Patent No. 568,392). Examples of MVA virus strains deposited in compliance with the requirements of the Budapest Treaty are strains MVA 572, MVA 575, and MVA-BN deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury (UK) with the deposition numbers ECACC V94012707, ECACC V00120707 and ECACC V00083008, respectively, and described in U.S. Pat. Nos. 7,094,412 and 7,189,536.
MVA-BN® is a virus used in the manufacturing of a stand-alone third generation smallpox vaccine. MVA-BN® was developed by further passages from MVA strain 571/572. To date, more than 1800 subjects including subjects with atopic dermatitis (AD) and HIV infection have been vaccinated in clinical trials with MVA-BN® based vaccines. The renewed interest in smallpox vaccine-campaigns with Vaccinia-based vaccines has initiated an increased global demand for large-scale smallpox vaccine production. Furthermore, the use of poxviruses as a tool for preparation of recombinant vaccines has additionally created significant industrial interest in methods for manufacturing (growth and purification) of native Vaccinia viruses and recombinant-modified Vaccinia viruses.
Cell lines have become a valuable tool for vaccine manufacturing. The production of some important vaccines and viral vectors is still done in embryonated chicken eggs or primary chicken embryo fibroblasts. Vaccinia Viruses-based vaccines have in general been manufactured in primary CEF (Chicken Embryo Fibroblasts) cultures. Vaccines manufactured in primary CEF cultures are generally considered safe as regards residual contaminants. First, it is scientifically unlikely that primary cell cultures from healthy chicken embryos should contain any harmful contaminants (proteins, DNA). Second, millions of people have been vaccinated with vaccines manufactured on CEF cultures, in accordance with various reports so far without any severe adverse effects resulting from the contaminants (CEF proteins and CEF DNA). There is, therefore, no regulatory requirement for the level of host cell contaminants in vaccines manufactured in primary CEF cultures, but for each vaccine the manufacturer must document its safety. The regulatory concern for vaccines manufactured in primary CEF cultures relates to the risk of adventitious agents (microorganisms (including bacteria, fungi, mycoplasma/spiroplasma, my cobacteria, rickettsia, viruses, protozoa, parasites, TSE agent) that are inadvertently introduced into the production of a biological product.
Viruses used in the manufacturing of vaccines or for diagnostic purposes can be harvested and purified in several ways depending on the type of virus. Traditionally, purification of pox viruses including Vaccinia viruses and recombinant-modified Vaccinia viruses has been carried out based on methods separating molecules by means of their size differences. To enhance removal of host cell contaminants (e.g. DNA and proteins), in particular DNA, the primary purification by means of size separation has been supplemented by secondary methods such as enzymatic digestion of DNA (e.g. Benzonase treatment). Most commonly, the primary purification of Vaccinia viruses and recombinant-modified Vaccinia viruses has been performed by sucrose cushion or sucrose gradient centrifugation at various sucrose concentrations. Recently, ultrafiltration has also been applied either alone or in combination with sucrose cushion or sucrose gradient purification.
In the current methods for purification of Vaccinia viruses, manufactured in primary CEF culture the level of CEF protein may be up to 1 mg/dose and the CEF DNA level may exceed 10 μg/dose of 1×108 as measured by the TCID50. These levels are considered acceptable from a safety and regulatory perspective as long as the individual vaccine manufacturer demonstrates that the levels to be found in the Final Drug Product (FDP) are safe at the intended human indications. Due to the risk of presence of adventitious agents in vaccines manufactured in primary cell cultures and the associated need for extensive, expensive biosafety testing of each vaccine batch manufactured, there is a strong stimulus for the vaccine industry to change to continuous cell lines. Once a continuous cell line has been characterized the need for testing for adventitious agents of the production batches is minimal.
However, switching from primary to continuous cell culture for production of Vaccinia and Vaccinia recombinant vaccines is expected to impose stricter safety and regulatory requirements. In fact, the regulatory authorities have proposed new requirements for levels of DNA contaminants in vaccines manufactured using continuous cell lines (See Draft FDA guideline), which may be as low as 10 ng host-cell DNA/dose. To achieve such low level of host cell contaminants, new and improved methods for harvesting and purification are needed.
Thus far, host cells (in particular, CEF cells) for poxviruses are typically cultured in roller flasks (also known as roller bottles) or in cell factories (such as disposable fixed-bed bioreactors). Roller flasks are cylindrical screw-capped flasks mostly made of disposable plastic; reusable glass ones are still used occasionally. Each flask is typically about 1 to 1.5 liters in total volume. Typically, a flask is filled with 0.1 to 0.3 liters of culture medium for cell cultivation. A stack of flasks is placed on a roller, the flasks rotate on the roller rack at 1 to 4 rpm and are incubated in an incubator or an incubation room. Roller flasks are used for the cultivation of both suspension cells and adherent cells. Roller flasks are only used in small scale when, for example, convenience and/or an aseptic production dictates this selection of cultivation methods. However, since culturing in roller flasks is cost intensive, tedious and not readily and conveniently scalable by keeping GMP-principles of aseptic processing to large scale production, manufacturers sought and are still seeking alternatives.
As an alternative to cultivation in roller flasks, host cells for poxviruses can also be grown by the use of the WAVE Bioreactor™ system. This system is a cell culture device suitable for applications in animal, virus, insect, and plant cell culture in suspension, or on microcarriers, as well as cellular therapeutics. The WAVE Bioreactor™ system consists of two components: disposable cell bags and a rocker. Culture medium and cells only contact a presterile, disposable chamber called a cellbag that is placed on a special rocking platform. The rocking motion of this platform induces waves in the culture fluid. These waves provide mixing and oxygen transfer, resulting in a perfect environment for cell growth that can easily support over 10×106 cells/ml.
Typically, when grown in roller flasks, cells adhere to the walls of the roller flask. The cells can be detached from the walls, for example, mechanically or enzymatically (such as trypsinization). Subsequently, host cells can be subjected to ultrasound treatment or high-pressure homogenization to obtain a homogenate (see WO 2003/054175) which can be further purified.
Alternatively, in order to release viruses from their host cells, culture medium is discarded (poured off) from roller flasks and cells are lysed, typically by way of a hypotonic lysis buffer. Subsequently, the mixture of lysed cells and viruses released from said cells are subject to filtration in order to obtain the released viruses. Thus, cell lysis takes place in the roller flasks. One can imagine that the afore-described process is highly susceptible to become non-aspectic, since roller flasks have to be opened to discard culture medium and to add cell lysis buffer. After that, they have to be re-opened once more in order to then obtain the released viruses by filtration such as depth filtration (see WO 2006/052826, in particular Example 2).
Though culturing host cells for poxviruses in cellbags of the WAVE Bioreactor™ system is attractive, since the culture can be conveniently kept aseptic and the process is readily scalable, the process of harvesting and purifying poxviruses may be somewhat inconvenient and thus not desirable insofar that potentially measures non-optimal for aseptic processing may have to be taken such as batch centrifugation or flow-through centrifugation of the host cells in order to harvest them. This is so because, in cellbags host cells do not grow adherently, but in suspension and, thus, culture medium cannot easily be discarded prior to cell lysis in order to release viruses. Accordingly, cells must be separated and thereby concentrated. However, batch centrifugation includes open process steps which are non-optimal for aseptic processing. Flow-through centrifugation, on the other hand, might not remove the complete cell culture medium, because the cells have to remain suspended to be able to remove them from the flow-through centrifuge at the end of the centrifugation. If cell culture medium is not removed completely, residual salt content might impair efficiency of subsequent processes, such as a subsequent hypotonic lysis step which probably results in a final product with insufficient yield (e.g. virus titer) and elevated impurity level.
Thus, in essence, in order to produce expression products such as therapeutic proteins or viruses for vaccination purposes under GMP standards including aseptic processing in large (industrial) scale, it is desirable to grow host cells in suspension in, for example, cellbags because of their advantageous properties (e.g., ideal aseptic environment for cell growth in high density culture). However, cells in suspension (e.g. in cellbags) may be disadvantageous, since harvesting and disrupting host cells may not be practicable in a closed process which retains an optimal yield.
Accordingly, there is a need for means and methods to harvest host cells, preferably grown under GMP and principles of aseptic processing, preferably in a closed process and to then release their intracellular or cell-associated expression product, also under said GMP-principles, and concomitantly obtaining the maximum yield of said expression product, preferably in the absence of impurities such as cell debris and/or culture medium. Hence, it is an aim of the present invention to comply with these needs and to thus provide a solution to the existing problem.